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Murine gammaherpesvirus 68 (HV68) infection of mice results in the establishment of a chronic infection, which is largely maintained through latent infection of B lymphocytes. Acute virus replication is almost entirely cleared by 2 weeks postinfection. Spontaneous reactivation of HV68 from latently infected splenocytes upon ex vivo culture can readily be detected at the early stages of infection (e.g., day 16). However, by 6 weeks postinfection, very little spontaneous reactivation is detected upon explant into tissue culture. Here we report that stimulation of latently infected splenic B cells harvested at late times postinfection with cross-linking surface immunoglobulin (Ig), in conjunction with anti-CD40 antibody treatment, triggers virus reactivation. As expected, this treatment resulted in B-cell activation, as assessed by upregulation of CD69 on B cells, and ultimately B-cell proliferation. Since anti-Ig/anti-CD40 stimulation resulted in splenic B-cell proliferation, we assessed whether this reactivation stimulus could overcome the previously characterized defect in virus reactivation of a v-cyclin null HV68 mutant. This analysis demonstrated that anti-Ig/anti-CD40 stimulation could drive reactivation of the v-cyclin null mutant virus in latently infected splenocytes, but not to the levels observed with wild-type HV68. Thus, there appears to be a role for the v-cyclin in B cells following anti-Ig/ anti-CD40 stimulation independent of the induction of the cell cycle. Finally, to assess signals that are not mediated through the B-cell receptor, we demonstrate that addition of lipopolysaccharide to explanted splenocyte cultures also enhanced virus reactivation. These studies complement and extend previous analyses of Epstein-Barr virus and Kaposi’s sarcoma-associated virus reactivation from latently infected cell lines by investigating reactivation of HV68 from latently infected primary B cells recovered from infected hosts. Background. Gammaherpesviruses are characterized by their ability to establish latency in lymphocytes. The establishment and maintenance of latency is important for persistence in the infected host, while virus reactivation is needed for transmission of the virus to new hosts and may also be required to maintain reservoirs of latently infected cells in the chronically infected host (2, 12, 15, 33, 34). The switch between latency and the lytic cycle for the human gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi’s sarcoma-associated herpesvirus (KSHV), has been extensively characterized in established latently infected cell lines in vitro (11, 27, 42). Initiation of the EBV lytic cycle can be stimulated by several different reagents, including anti-immunoglobulin (anti-Ig), calcium ionophore, sodium butyrate, and the phorbol ester tetradecanoyl phorbol acetate (TPA) (27). KSHV reactivation can also be induced by stimulation with phorbol esters and sodium butyrate (3, 19).

The gammaherpesvirus immediate-early genes are critical regulators of virus replication and reactivation from latency. Rta, encoded by gene 50, serves as the major transactivator of the lytic program and is highly conserved among all the gammaherpesviruses, including Epstein-Barr virus, Kaposi's sarcoma-associated herpesvirus, and murine gammaherpesvirus 68 (γHV68). Introduction of a translation stop codon in γHV68 gene 50 (gene 50.stop γHV68) demonstrated that Rta is essential for virus replication in vitro. To investigate the role that virus replication plays in the establishment and maintenance of latency, we infected mice with gene 50.stop γHV68. Notably, the gene 50.stop virus established a long-term infection in lung B cells following intranasal infection of mice but was unable to establish latency in the spleen. This complete block in the establishment of latency in the spleen was also seen when lytic virus production was inhibited by treating mice infected with wild-type virus with the antiviral drug cidofovir, implicating virus replication and not an independent function of Rta in the establishment of splenic latency. Furthermore, we showed that gene 50.stop γHV68 was unable to prime the immune system and was unable to protect against a challenge with wild-type γHV68, despite its ability to chronically infect lung B cells. These data indicate gammaherpesviruses that are unable to undergo lytic replication in vivo may not be viable vaccine candidates despite the detection of cells harboring viral genome at late times postinfection.

Murine gammaherpesvirus 68 (γHV68) provides a tractable small animal model with which to study the mechanisms involved in the establishment and maintenance of latency by gammaherpesviruses. Similar to the human gammaherpesvirus Epstein-Barr virus (EBV), γHV68 establishes and maintains latency in the memory B-cell compartment following intranasal infection. Here we have sought to determine whether, like EBV infection, γHV68 infection in vivo is associated with B-cell proliferation during the establishment of chronic infection. We show that γHV68 infection leads to significant splenic B-cell proliferation as late as day 42 postinfection. Notably, γHV68 latency was found predominantly in the proliferating B-cell population in the spleen on both days 16 and 42 postinfection. Furthermore, virus reactivation upon ex vivo culture was heavily biased toward the proliferating B-cell population. DNA methyltransferase 1 (Dnmt1) is a critical maintenance methyltransferase which, during DNA replication, maintains the DNA methylation patterns of the cellular genome, a process that is essential for the survival of proliferating cells. To assess whether the establishment of γHV68 latency requires B-cell proliferation, we characterized infections of conditional Dnmt1 knockout mice by utilizing a recombinant γHV68 that expresses Cre-recombinase (γHV68-Cre). In C57BL/6 mice, the γHV68-Cre virus exhibited normal acute virus replication in the lungs as well as normal establishment and reactivation from latency. Furthermore, the γHV68-Cre virus also replicated normally during the acute phase of infection in the lungs of Dnmt1 conditional mice. However, deletion of the Dnmt1 alleles from γHV68-infected cells in vivo led to a severe ablation of viral latency, as assessed on both days 16 and 42 postinfection. Thus, the studies provide direct evidence that the proliferation of latently infected B cells is critical for the establishment of chronic γHV68 infection.